The F0F1-ATP synthase is responsible for synthesizing most of the ATP consumed by mammalian cells. The synthase couples proton electrochemical gradients generated by electron transport to the condensation of ADP with Pi in the inner membrane of the mitochondrion. The F0F1-ATP synthases of mitochondria, plasma membranes of bacteria and thylakoid membranes of chloroplasts are similar in structure and synthesize and hydrolyze ATP by identical mechanisms. The F0 sector is a transmembrane protein complex that medlates proton conduction, whereas F1 is a peripheral protein complex that is attached to F0 by a narrow stalk. It contains the catalytic sites for ATP ysnthesis. When removed from the F0 as a soluble complex, F1 is an ATPase. In addition to three catalytic sites, the F1 ATPases contain three other nucleotide binding sites, the function of which has not been established. The overall aim of this collaborative project is to establish the functional role of the noncatalytic nucleotide binding sites in the F0F1-ATPases. The specific aims of the project are: I) to determine if the binding of ADP opposed to ATP to noncatalytic sites leads to different modulations of the ATPase, ATP synthase and proton conducting activities of the F0F1 complex; and 2) to determine if activating anions stimulate ATPase activity by binding to noncatalytic sites or to sites located in the pathway for cross-talk between noncatalytic sites and catalytic sites. The CF0F1 complex of chlorplast thylakoid membranes will be used in Dr. Malyan's laboratory to accomplish specific aim 1). This system can be easily manipulated to provide coupled member vesicles in which the noncatalytic sites of the CF1 component are either free of nucleotides or loaded with 2 mol of ADP or ATP per moL The majority of the experiments designed to accomplish specific aim 2) will also be carried out in Dr. Malyan's laboratory using the CF0F1 ATP synthase in thylakoid vesicles. However, a complimentary experimental program will be conducted in Dr. Allison's laboratory which is designed to provide information on the binding site for activating anions using mutants of the alpha3beta3gamma3 subcomplex of the TF1-ATPase isolated from the thermophillic Bacillus PS3. The latter are continuing experiments supported by the parent grant.